Human gastrointestinal disease can be caused by a variety of microorganisms. Common vehicles for infection are contaminated foods and water. To reduce the incidence of such disease, foods and water destined for human consumption are routinely tested for their sanitary quality. Instead of testing for a multitude of different enteric pathogens, laboratories test for the presence of indicator organisms. Coliform bacteria are typically used as the primary indicators of sanitary quality because they are commonly associated with the gastrointestinal tracts of warm-blooded animals. The presence of coliform bacteria, especially Escherichia coli, in high numbers in foods or water suggests that there may be fecal contamination, and contraindicates human consumption.
Coliform bacteria are distinguished from other organisms and from their close relatives in the family Enterobacteriaceae by their ability to ferment lactose to acidic and gaseous (CO.sub.2 and H2) end products. Certain non-coliform bacteria may ferment lactose to the same end products, but the growth of these organisms in the coliform assay is usually minimized or prevented by the selective properties of the bacteriological media used for testing.
Typically, the presence of coliform bacteria in a sample is determined by adding the material to a liquid bacteriological growth medium and incubating the mixture at a temperature which is conducive to bacterial growth. Incubation of the mixture of sample and growth medium is important because the assay must detect low levels of coliform bacterial contamination in the sample. Incubation of the mixture results in the multiplication of coliforms to a level of approximately 10.sup.6 to 10.sup.8 cells per milliliter of culture, wherein their presence may be detected by any of a number of techniques. The variety of different liquid bacteriological growth media which have been used for coliform detection share two common properties: they contain the disaccharide lactose and they also contain chemical agents which selectively inhibit the growth of non-coliform microorganisms. Selection is important because the sample invariably contains a variety of microorganisms, and the success of the assay depends on the coliforms not being overgrown by the non-coliforms.
Coliform assays can be performed using either solid or liquid media. Assays which employ a solid medium allow a viable cell count. Sample is added to the solid medium and discrete colonies of coliforms are enumerated. Alternatively, samples may be added to liquid media. Coliforms are detected through the formation of characteristic metabolic end products. The liquid medium format may be qualitative or quantitative. The liquid medium format is preferred for samples containing fewer coliforms (e.g., less than 10 organisms per milliliter), or samples containing particulate material (e.g., food or dairy samples) which obscures colony visualization.
Most coliform assays in a broth or agar take place in two discrete stages: presumptive and confirmed. First, a presumptive assay provides an indication of possible coliform presence. In the confirmed stage, presumptively positive cultures or typical colonies are subcultured into a second, more selective medium. In principle, the confirmed medium eliminates false positive results. Together, the two stages of the assay require 48 to 96 hours for completion. Therefore, there is a need in the art to provide accurate results in a more timely manner.
Generally, detection is based upon the end products of metabolic pathways of coliform bacteria. For example, acid production by coliform bacteria is generally detected through the incorporation of pH indicators in the medium. Acid formation can be detected by a change in color from purple to yellow of the indicator bromocresol purple in a liquid medium such as Clarke's medium. Acid production is detected by the formation of colonies which are dark-centered due to the reaction between the acid and the indicator neutral red in a solid medium such as violet red bile agar (VRBA) and MacConkey agar.
Further, gaseous end products are usually detected by the presence of gas bubbles in a liquid. Gas bubbles may be entrapped in a smaller, inverted test tube or an inverted vial within the culture tube or in a special portion of the culture device. For example, the BioControl ColiTrak.TM. product entraps gas bubbles in a dome associated with the top portion of the device. Petrifilm.TM. (3M), with a solid growth medium, entraps gas bubbles in close proximity to a bacterial colony. Alternatively, gaseous end products may be detected by nonvisual means such as by electrochemical detection of hydrogen, by radiometric detection of .sup.14 CO.sub.2 released from the fermentation of radiolabelled lactose, or by impedimetric or gas chromatographic detection of organic compounds produced during fermentation.
An alternative approach to coliform detection is based upon the detection of coliform-associated enzyme activity rather than metabolic end products. Generally, an enzyme assay approach can yield quantitative estimates for coliform bacteria comparable to confirmatory results using lengthy culture testing procedures. The enzyme beta-galactosidase is a bacterial enzyme used for the fermentation of lactose. Beta-galactosidase hydrolyzes lactose to its component sugars glucose and galactose. Coliform bacteria typically express this enzyme. For example, Warren et al., Appl. Environ. Microbiol. 35:136-41, 1978, refers to a coliform testing method which uses the chromogenic beta-galactosidase substrate, o-nitrophenyl-beta-D-galactoside (ONPG), for quantitating fecal coliforms in water. In Warren et al., the time of appearance of the yellow reaction product o-nitrophenol (ONP) is inversely related to the initial concentration of coliforms in the test sample.
The product Colilert.TM. (Access Medical) uses the ONPG enzyme indicator for detecting coliforms in drinking water. The water sample is used to solubilize a basal growth medium containing ONPG. Beta-galactosidase from the bacteria growing in the culture hydrolyzes the ONPG to produce galactose, which serves as the sole carbon and energy source for growth. ONP formation indicates enzyme activity. After 24 hours of incubation, the presence of a yellow color throughout the liquid culture of 10 milliliters signifies that coliform bacteria were present in the water sample.
The fluorogenic beta-galactosidase substrate fluorescein-beta-D-galactoside is another indicator of activity. Cundell et al., Proc. Water Reuse Symp. 3:1895-99, 1979, refers to an assay incubating coliform bacteria in a medium containing the fluoresceinobeta-D-galactoside. Coliform bacteria are determined quantitatively by flow cytometry. The fluorescein moiety of the substrate becomes concentrated within the cell after cleavage by beta-galactosidase and imparts fluorescence to the cells under ultraviolet illumination. U.S. Pat. No. 4,242,447 refers to a fluorescent assay for enumerating coliforms using a fluorogenic substrate. Specifically, U.S. Pat. No. 4,242,447 refers to a process emulsifying a water sample and a fluorogenic substrate with an oil to form oil droplets containing coliform bacteria. Beta-galactosidase activity cleaves the fluorogenic substrate and forms a fluorescent oil droplet which is counted in a fluorescence detector. Cundell et al., Dev. Ind. Microbiol. 20:571-77, 1979, refers to a similar technique that yields oil-encapsulated cells on a microscope slide. Fluorescent droplets are counted under a fluoresence microscope. The Cundell method has been used with sewage samples. The sensitivity of the Cundell method is 10.sup.5 cells per milliliter.
The bacterium, E. coli, is a coliform which may be assayed separate and apart from the coliform bacteria. E. coli presence is considered to be a more reliable indicator of fecal contamination. Additionally, certain strains of E. coli are pathogenic for humans and animals. The standard E. coli detection method subcultures positive presumptive cultures into EC broth and incubates at 45.5.degree. C. Gas-positive EC cultures are then streaked onto a differential agar medium. The isolates are identified by biochemical characterization.
E. coli is a relatively unique organism by possessing the enzyme beta-glucuronidase. Consequently, the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) is used to detect E. coli in food and water samples. The E. coli detection procedure consists of inoculating cultures and incubating by standard methods in the presence of MUG. Fluorescence which develops during 24 hours incubation indicates the presence of E. coli because beta-glucuronidase from E. coli cleaves MUG to a fluorescent product. Thus, the use of MUG-containing media can shorten the detection time for E. coli. Unfortunately, MUG is an expensive reagent and large sample sizes, such as water samples, require large quantities of MUG and make the test prohibitively expensive. Currently, the recommended concentration of MUG is 50-100 micrograms per milliliter of final culture. Typical volumes for food and water tests are 90 and 100 milliliters respectively. Thus, the cost of the MUG reagent makes it unattractive for food testing and for use in the PA type of water testing.
One can assay for beta-galactosidase and beta-glucuronidase activities as a means for determining the presence of coliforms and E. coli, respectively. Enzyme detection assays offer advantages over the detection of fermentation end products. First, the enzyme assay approach is more sensitive. One enzyme can cleave many substrate molecules. Each molecule of substrate cleaved by an enzyme yields a fluorescent or colored reporter product. Therefore, the signal is amplified by an enzyme. With certain substrates, as few as 10.sup.4 metabolically active cells can yield a signal within 24 hours.
By contrast, it takes approximately 10.sup.7 to 10.sup.8 metabolically active cells to produce a visible gas bubble within 24 hours, because beta-galactosidase cleavage of lactose does not result in stoichiometric amounts of acid and gas end products due to physiological regulation of metabolism. The majority of the carbon in the lactose molecule is incorporated into cellular biomass and is not used to regenerate oxidized AND during fermentation. The bacterial fermentation enzymes responsible for acid and gas production are at the end of the fermentation pathway and are tightly regulated. For instance, pyruvate-formate lyase is inactive under aerobic conditions and is activated only when the culture becomes anaerobic. Formate dehydrogenase, a key enzyme in gas production, is not synthesized in the presence of oxygen or when the culture pH is 6 or greater. Hydrogenase activity and synthesis are negatively regulated by alternative oxidants such as oxygen, nitrate and nitrite. Accordingly, it is possible that growth conditions of anaerobiosis, acidic pH and the absence of external oxidants are either not achieved or are achieved only slowly. Moreover, much of the lactose in the culture will have been cleaved by the time the acid- and gas-producing enzymes become active. Therefore, enzyme assays for beta-galactosidase circumvent the fermentation pathway regulation problems and yield a reporter group with every substrate cleavage event.
The presence of anaerogenic strains of genera which belong to the coliform group further complicates gas detection assay procedures. These strains possess beta-galactosidase activity, but do not produce gas from lactose. Thus, only an assay for beta-galactosidase activity would detect the anaerogenic strains of the coliform group.
It is possible to detect coliform-associated enzymes within 24 hours of incubation. Beta-galactosidase and beta-glucuronidase assay results correlate with the confirmed presence of coliforms and E. coli. When aliquots from 24 hour enzyme assay-positive coliform cultures are streaked onto a differential medium, such as eosin methylene blue agar, and the agar incubated, typical coliform and E. coli colonies are almost always recovered. Members of the coliform genera are the taxonomically identified colonies from beta-galactosidase-positive cultures. E. coli is the taxanomically identified organism from beta-glucuronidase-positive cultures. These observations indicate that the first incubation (presumptive) stage of the standard coliform assay can be shortened from 48 to 24 hours, and that the second (confirmed) incubation stage is unnecessary. Consequently, enzyme assays for coliforms and E. coli can reduce the time required to produce a confirmatory result by as much as 72 hours, and simultaneously, provide significantly better sensitivity. However, the cost is more expensive due to the reagents, especially for large sample sizes.
Food and dairy samples present problems detecting coliforms. There is a large diversity of foods and the physical/chemical nature of some makes the detection of either acidic or gaseous end products or certain types of enzyme assays difficult. For example, gas bubbles may be difficult to detect in a broth due to the turbidity from a food sample such as nonfat dried milk. The color associated with a food product such as dried cheese, chili powder or green or yellow vegetables can obscure the color of a pH indicator or a colored reporter, such as ONP, in a beta-galactosidase assay using ONPG as a substrate. Further, a food may be inherently acidic, or it may internally trap gas bubbles during sample preparation. Finally, food chemistry may interfere with an assay. Foods add additional nutrients to a culture broth and these nutrients may be metabolized by non-coliform bacteria to produce gaseous or acidic end products leading to a false-positive result. For example, cake mixes or other sweetened products contain significant amounts of disaccharides and monosaccharides, which can be converted to acid and/or gas by non-coliforms.
It is important to choose an appropriate substrate for beta-galactosidase and beta-glucuronidase assays for coliform bacterial and E. coli detection. For example, beta-galactosidase cleaves the substrate ONPG to a yellow product, ONP. However, certain colors inherent in or added to food products may obscure the yellow color of the ONP reporter product. Food substances which can obscure the yellow color include hemoglobin in red meats, chlorophyll in leafy greens and vegetables, beta-carotene and other yellow and orange pigments in fruits and vegetables, natural pigments in spices, and artificial and natural colorants. Turbid water and water with a high humic content can also obscure weakly yellow positive ONP reactions. Moreover, standard bacteriological growth media ingredients, such as protein or yeast hydrolysates, impart a yellow Or golden color to the media and can obscure the presence of ONP. For example, the standard method coliform medium, lauryl sulfate tryptose (LST) broth, has a golden hue. The result of performing an ONPG-type enzyme assay in LST broth and other supplemented media will lead to a significant number of false-negative results.
An additional problem associated with the use of ONPG is the hazardous nature of the ONP cleavage product. ONP is a volatile compound and a known respiratory, eye and skin irritant. The presence of ONP makes activities associated with handling of cultures and washing of glassware hazardous to lab workers. There are also toxic waste disposal problems when using ONP.
Similarly, beta-glucuronidase assays use MUG, which generates a fluorescent signal. Pigments associated with foods have been observed to quench the fluorescent signal. For example, the pigment in chili powder obscures the fluorescence reaction of the reporter product. A further problem with MUG is that the pH range for maximal fluorescence is outside of the pH range produced during lactose fermentation. MUG is maximally fluorescent above pH 10. Yet, acids produced from lactose fermentation typically lower the coliform culture medium to pH 5 to 6, obscuring fluorescence reactions. Finally, as noted above, beta-glucuronidase assays which employ chromogenic substrates may be subject to interference from some types of samples. Therefore, current enzyme assays for beta-glucuronidase have pH and interference problems from the food samples, and are inappropriate for use in certain types of foods.
Fluorogenic substrates, such as MUG have been used in agar to detect E. coli in food samples. The fluorescent cleavage product, 4-methylumbelliferone, is soluble and will diffuse from E. coli colonies in agar (a solid medium) to add to the difficulties associated with the determination of which colonies contain the enzyme beta-glucuronidase and which do not.
An additional complication is that the final volume of culture broth for different types of samples varies from 1 ml to 1 liter. For each test, the molarity of the chromogenic or fluorogenic substrate within the culture medium would need to be constant. This would require consumption of large amounts of expensive test reagents. It would be advantageous to a food processor if all coliform testing could be done in a single assay format which features easily read results across all test samples, regardless of size, and which economizes on test reagents.
A typical food processing company or water treatment facility tests incoming and outgoing foods, process water, waste water or environmental samples. Therefore, an ONPG- or MUG-based assay may only be appropriate for certain types of samples. Thus, there is a need in the art for assays which are functional for a variety of samples, such as foods, turbid and/or colored water and environmental samples and which eliminate the difficulties associated with correlating different test methodologies.
The indigogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) has been used in solid media for the detection of E. coli colonies which express beta-galactosidase activity. Beta-galactoside cleaves X-Gal to galactose and the indolyl derivative. The indolyl derivative dimerizes to form a substituted indigo, which has an intense blue color.
Frampton et al., J, Food Prot. 51:402-04, 1988, refers to a peptone-tergitol agar supplemented with 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-Gluc) to differentiate E. coli from other bacterial colonies in artificially inoculated raw minced chicken. Ley et al., Ann. Meet., Am. Soc. Microbiol., Abstract Q35:288, 1988, refers to a method to enumerate E. coli in water and sewage using a membrane filtration technique with a glycerol medium and 3-indolyl-beta-D-glucuronide substrate. E. coli colonies appeared blue in the semisolid medium. Watkins et al., Appl, Environ. Microbiol, 54:1874-75, 1988, refers to a pour plate method using X-Gluc for detecting E. coli in shellfish and waste water.
A product Petrifilm.TM. (3M) uses a solid medium and X-Gluc for detecting E. coli. E. coli colonies appear blue.
Because of the high cost of the indogenic compounds X-Gal and X-Gluc, for the detection of beta-galactosidase and beta-glucuronidase activity have not found widespread use or have been used with relatively small culture volumes. Therefore, there is a need in the art for a beta-galactosidase test system that can economically use small quantities of X-Gal and X-Gluc for food and water testing, because the intense blue color will solve many of the problems associated with the yellow color of ONP and other colored reporters.
In summary, there is a need in the art, therefore, for a coliform detection assay which offers the improved sensitivity and speed associated with enzyme assays, the capacity to detect anaerogenic coliforms, successful applications to food, water and environmental samples, safety of handling and disposal, and economy in the consumption of expensive test reagents.